PROJECT SUMMARY Drug addiction is a chronic, relapsing disease characterized by compulsive drug seeking and use, despite harmful consequences. More specifically, opioid use disorder (OUD), often stemming from the misuse of prescription opioid painkillers, represents an urgent social and health crisis, responsible for approximately 50,000 yearly overdose deaths and incurring an annual burden of $78.5B in medical treatment, lost productivity and legal costs in the US (See NIDA website). While effective treatments exist for opioid overdose events, the lack of effective therapeutics for long-term abstinence and prevention of relapse highlight the pressing need for alternative approaches to study OUD. Moving beyond their direct effect on neuronal function, opioids are also known to act directly on microglia. Microglia are resident immune cells in the brain that serve as key drivers of neuroinflammation, a physiological process aimed at restoring homeostasis typically in response to a traumatic, chemical or ischemic insult to the central nervous system. A hallmark of neuroinflammation is microglial activation, and the transcriptional mechanisms underlying microglial activation operate under control of epigenetic remodeling of histone proteins. In the context of OUD, it has been previously shown that opioids can induce activation of microglia and that reduction in neuroinflammation can curb craving for opioid painkillers. Considering the known role of epigenetics in addiction and the emerging role of microglia in this disease, we propose to investigate the epigenetic underpinnings of microglial activation in OUD. More specifically, we will focus on the changes in histone lysine methylation in microglia of the brain reward system and how these changes comport with transcriptional programs across various phases of opioid- taking (maintenance, early withdrawal, and craving) by using a mouse model of intravenous remifentanil self-administration (IVSA). Furthermore, we will explore the role of this epigenetic modification in regulating opioid-induced microglial activation, opioid-seeking and relapse by pharmacological and genetic manipulation of Kdm6b, a histone lysine demethylase enriched in microglia. In conclusion, results from these experiments have the potential to not just broaden our understanding of the epigenetic mechanisms underlying OUD, but rather push the field of addiction epigenetics beyond the neuron and into cell types that could yield exciting new therapeutic avenues for the treatment this devastating disease.